Investigation of the Effects of Oral Nicotine Intake on Skin in Nicotine-Preferring Rat Lines
Project no. 32289 · Status: Kapanmış (Yayın bekleniyor) · 2024
Summary
<p><strong>INTRODUCTION AND OBJECTIVES:</strong> Nicotine, one of the most widely used addictive substances worldwide, is known to impair the synthesis/degradation balance of proteins such as collagen and elastin that maintain the structural integrity of the skin, thereby leading to premature skin aging, vascular damage, increased inflammatory response, and impaired tissue regeneration. However, individual differences in susceptibility to nicotine addiction and the underlying genetic/molecular framework of nicotine's dermatological effects have not been fully elucidated. The "Nicotine-Preferring Rat Line" (NP), developed through selective breeding for 31 generations at Ege University (EGE HAYMER), provides a novel model to investigate genetic susceptibility and molecular mechanisms in nicotine addiction.</p><p>The main objectives of this project are:</p><ul><li><ul><li>To determine the histopathological and molecular effects of chronic oral nicotine intake on skin tissue in the selectively bred nicotine-preferring rat line (NP),</li><li>To examine the effects of oral nicotine consumption on dermal vascularization (VEGF) as well as oxidative and inflammatory processes (iNOS, eNOS),</li><li>To compare nicotine-preferring rats that have never been exposed to nicotine (received pure water) with naive control animals, in order to reveal whether the genetic background/phenotypic predisposition conferring susceptibility to nicotine addiction leads to baseline alterations in skin structure.</li></ul></li></ul><p><strong>METHODS:</strong></p><ul><li><ul><li><strong>Experimental Groups and Study Design:</strong> A total of 40 male rats, aged 3–4 months, obtained from Ege University Laboratory Animals Application and Research Center, were used in the study: 20 naive Sprague-Dawley (KE) and 20 nicotine-preferring line (TE) rats. Male subjects were chosen to eliminate sex hormone variability. Following 1 week of acclimatization and 1 week of cage/handling habituation, the animals were housed individually and divided into 4 groups (n=10/group):</li><li><strong>Group 1 (KE + Water):</strong> Naive control + Pure water only</li><li><strong>Group 2 (TE + Water):</strong> Nicotine-preferring line + Pure water only</li><li><strong>Group 3 (KE + Nicotine):</strong> Naive control + Oral nicotine choice via two-bottle choice method</li><li><strong>Group 4 (TE + Nicotine):</strong> Nicotine-preferring line + Oral nicotine choice via two-bottle choice method</li><li><strong>Oral Nicotine Administration (Two-Bottle Choice Method):</strong> Animals in the nicotine-treated groups were subjected to a 24-hour free-access two-bottle choice protocol for 5 weeks. One bottle contained pure water, while the other contained water supplemented with nicotine 10mg/L for the first 2 weeks, 20mg/L for the remaining 3 weeks). Control groups received pure water in both bottles.</li><li><strong>Sacrifice and Tissue Sampling:</strong> At the end of the 5-week intervention period, animals were euthanized by decapitation. A 2x3cm area of thin skin on the dorsal region was shaved, and 1cm full-thickness skin biopsy samples were excised and immediately immersed in 4% paraformaldehyde (PFA) fixative.</li><li><strong>Histopathological and Immunohistochemical (IHC) Analyses:</strong> Fixed tissue samples were processed through routine tissue processing steps (dehydration, clearing, paraffin embedding) and blocked. Sections of 5 um thickness were cut using a microtome.</li><li><em>Histochemical Evaluation:</em> Hematoxylin-Eosin (H&E) staining was performed for overall tissue and cellular morphology; Masson’s Trichrome staining was used to analyze dermal collagen fiber organization and connective tissue alterations.</li><li><em>Immunohistochemical (IHC) Analysis:</em> Expression levels and localizations of VEGF (as a marker of local angiogenesis and vascularization) as well as iNOS and eNOS (as markers of oxidative and inflammatory processes) were evaluated under a light microscope.</li><li><strong>Statistical Analysis:</strong> Data were analyzed using SPSS v.25 software. Results are presented as "Mean \pm Standard Deviation". Appropriate parametric tests (including Bonferroni-adjusted post-hoc tests) were utilized for inter-group comparisons and time-dependent changes. A value of p < 0.05 was considered statistically significant.</li></ul></li></ul><p><strong>RESULTS:</strong></p><ul><li><ul><li><strong>Histopathological Findings (H&E and Masson’s Trichrome):</strong></li><li><strong>Group 1 (KE + Water):</strong> Dermal layers and collagen/elastin fiber structures were fully preserved.</li><li><strong>Group 2 (TE + Water):</strong> Despite having never received nicotine, the genetically predisposed line exhibited baseline mild disorganization in collagen fiber arrangement and a reduction in fiber density.</li><li><strong>Group 3 (KE + Nicotine):</strong> Oral nicotine consumption resulted in marked loosening, thinning, and degeneration of collagen fibers.</li><li><strong>Group 4 (TE + Nicotine):</strong> Extracellular matrix breakdown, fiber fragmentation, and degenerative changes were observed at the highest severity.</li><li><strong>Immunohistochemical Findings (VEGF, iNOS, and eNOS):</strong></li><li><strong>VEGF:</strong> Angiogenic response and neovascularization induction increased in parallel with nicotine intake, with the highest expression observed in the TE + Nicotine group (p < 0.05).</li><li><strong>iNOS and eNOS:</strong> Markers of oxidative stress and inflammatory response were markedly elevated in nicotine-treated groups. The highest immunoreactivity and endothelial stress were observed in the TE + Nicotine group (p < 0.05).</li></ul></li></ul><p><strong>DISCUSSION:</strong></p><p>The primary objective of this study was to investigate the histopathological and molecular effects of oral nicotine intake on dermal tissue in our selectively bred nicotine-preferring rat line (NP/TE), and to evaluate whether the genetic background conferring addiction vulnerability creates baseline phenotypic differences in the skin.</p><p>Key findings and evaluations are as follows:</p><ul><li><ul><li><strong>Dermal Matrix Degradation:</strong> Masson’s Trichrome and H&E analyses demonstrated that chronic oral nicotine intake compromises extracellular matrix integrity by inducing pronounced loosening, thinning, and degeneration in dermal collagen fiber bundles.</li><li><strong>Induction of Vascular and Oxidative Stress:</strong> Immunohistochemical (IHC) data revealed that nicotine significantly upregulates the expression of <strong>VEGF</strong>—an indicator of cutaneous hypoxia or altered angiogenesis—as well as <strong>iNOS</strong> and <strong>eNOS</strong>, key markers of inflammatory and oxidative stress.</li><li><strong>Peripheral Manifestation of Genetic Predisposition (Most Novel Finding):</strong> Nicotine-preferring rats exposed solely to vehicle (pure water) without prior nicotine exposure (TE + Water) exhibited baseline collagen fiber disorganization and moderate elevations/alterations in iNOS, eNOS, and VEGF expressions compared to naive controls. This indicates that the genetic/epigenetic background conferring addiction vulnerability is not restricted to the central nervous system, but also imparts an inherent baseline susceptibility to peripheral skin tissue.</li></ul></li></ul><p><strong>Conclusion:</strong> Our study not only confirms the destructive effects of nicotine on the skin but also fills an important gap in the literature by demonstrating, for the first time in the world, the peripheral phenotypic manifestations of genetic addiction vulnerability.</p><p><em style="background-color: white; color: rgb(33, 37, 41);">Projects Commission (Grant:32289) and Animal Ethics Committee (EÜHADYEK 2024-012).</em></p>